In Arabidopsis, the DELLA subfamily of GRAS regulatory genes consists of GAI, RGA, RGA-LIKE1 (RGL1), RGL2, and RGL3. GAI and RGA are known to be negative regulators of gibberellin (GA) responses. We found that RGL1 is a similar repressor of GA responses, as revealed by RGL1 gain-of-function and loss-of-function phenotypes. Repression of GA responses in Arabidopsis was conferred by a dominant 35S-rgl1 transgene carrying a DELLA domain deletion analogous to the GA-insensitive gai-1 mutation. As in GA-deficient Arabidopsis, the transgenic plants were dark green dwarfs with underdeveloped trichomes and flowers. Expression levels of GA4, a feedback-regulated GA biosynthetic gene, were increased correspondingly. Conversely, a loss-of-function rgl1 line had reduced GA4 expression and exhibited GA-independent activation of seed germination, leaf expansion, flowering, stem elongation, and floral development, as detected by resistance to the GA biosynthesis inhibitor paclobutrazol. RGL1 plays a greater role in seed germination than do GAI and RGA. The expression profile of RGL1 differed from those of the four other DELLA homologs. RGL1 message levels were predominant in flowers, with transcripts detected in developing ovules and anthers. As with RGA, green fluorescent protein (GFP)–tagged RGL1 protein was localized to the nucleus, but unlike GFP-RGA, there was no degradation after GA treatment. These findings indicate that RGL1 is a partially redundant, but distinct, negative regulator of GA responses and suggest that all DELLA subfamily members might possess separate as well as overlapping roles in GA signaling.

Arabidopsis thaliana has five ethylene hormone receptors, which bind ethylene and elicit responses critical for plant growth and development. Here we describe a negative regulator of ethylene responses, REVERSION-TO-ETHYLENE SENSITIVITY1 ( RTE1 ), which regulates the function of at least one of the receptors, ETR1 , in Arabidopsis . RTE1 was identified based on the ability of rte1 mutations to suppress ethylene insensitivity of the dominant gain-of-function allele etr1-2 . rte1 loss-of-function mutants have an enhanced ethylene response that closely resembles the etr1 null phenotype. The etr1 rte1 double null mutant is identical to the etr1 and rte1 single null mutants, suggesting that the two genes act in the same pathway. rte1 is unable to suppress the etr1-1 gain-of-function allele, placing RTE1 at or upstream of ETR1 . rte1 also fails to suppress gain-of-function mutations in each of the four other ethylene receptor genes. RTE1 encodes a previously undescribed predicted membrane protein, which is highly conserved in plants and protists but absent in fungi and prokaryotes. Ethylene treatment induces RTE1 expression, and overexpression of RTE1 confers reduced ethylene sensitivity that partially depends on ETR1 . These findings demonstrate that RTE1 is a negative regulator of ethylene signaling and suggest that RTE1 plays an important role in ETR1 function.

The gaseous hormone ethylene is perceived in Arabidopsis by a five member receptor family that consists of the subfamily 1 receptors ETR1 and ERS1 and the subfamily 2 receptors ETR2, ERS2, and EIN4. Previous work has demonstrated that the basic functional unit for the ethylene receptor, ETR1, is a disulfide-linked homodimer. We demonstrate here that ethylene receptors isolated from Arabidopsis also interact with each other through noncovalent interactions. Evidence that ETR1 associates with other ethylene receptors was obtained by co-purification of ETR1 with tagged versions of ERS1, ETR2, ERS2, and EIN4 from Arabidopsis membrane extracts. ETR1 preferentially associated with the subfamily 2 receptors compared with the subfamily 1 receptor ERS1, but ethylene treatment affected the interactions and relative composition of the receptor complexes. When transgenically expressed in yeast, ETR1 and ERS2 can form disulfide-linked heterodimers. In plant extracts, however, the association of ETR1 and ERS2 can be largely disrupted by treatment with SDS, supporting a higher order noncovalent interaction between the receptors. Yeast two-hybrid analysis demonstrated that the receptor GAF domains are capable of mediating heteromeric receptor interactions. Kinetic analysis of ethylene-insensitive mutants of ETR1 is consistent with their dominance being due in part to an ability to associate with other ethylene receptors. These data suggest that the ethylene receptors exist in plants as clusters in a manner potentially analogous to that found with the histidine kinase-linked chemoreceptors of bacteria and that interactions among receptors contribute to ethylene signal output.

Arabidopsis (Arabidopsis thaliana) RTE1 encodes a membrane protein and negatively regulates ethylene responses. Genetic and transformation studies suggest that the function of the wild-type RTE1 is primarily dependent on ETR1 and can be independent on the other receptors. Ethylene insensitivity caused by the overexpression of RTE1 is largely masked by the etr1-7 mutation, but not by any other receptor mutations. The wild-type ETR1 N terminus is sufficient to the activation of the RTE1 function and the ectopic expression of etr1(1–349) restored ethylene insensitivity conferred by 35S∷gRTE1 in etr1-7. The RTE1 N terminus is not essential to the etr1-2 function and the expression of rte1(NΔ49), which has an N-terminal deletion of 49 amino acid residues, restored ethylene insensitivity in etr1-2 rte1-2. The ectopic expression of GREEN FLUORESCENT PROTEIN (GFP)-RTE1 conferred ethylene insensitivity in wild type and the GFP fusion displayed fast movement within the cytoplasm. The GFP-RTE1 and EYFP-NAG proteins colocalized and the Brefeldin A treatment caused aggregation of GFP-RTE1, suggesting RTE1 is a Golgi-associated protein. Our results suggest specificity of the RTE1 function to ETR1 and that endomembranes may play a role in the ethylene signal transduction.